Multi-focus optical-resolution photoacoustic microscopy with ultrasonic array detection

ABSTRACT

A probe for use with an imaging system, including a scanning device configured to receive a first light beam from a light source, a beam-divider configured to split the first light beam into a plurality of second light beams, and a focusing device configured to focus each of the second light beams on respective locations in an object of interest is disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of prior U.S. application Ser. No.13/369,558, filed on Feb. 9, 2012, the entirety of which is incorporatedherein in its entirety. U.S. application Ser. No. 13/369,558 claims thebenefit of U.S. Provisional Application No. 61/442,148 filed Feb. 11,2011, which is also incorporated herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under grants R01EB000712 and U54 CA136398, both awarded by the U.S. National Institutesof Health. The government has certain rights in the invention.

BACKGROUND

The ability to image microstructures, such as the micro-vascular networkin the skin, the GI tract, or the brain cortex, and to monitorphysiological functions of tissue is invaluable. One of the promisingtechnologies for accomplishing this objective is photoacousticmicroscopy. Current high-resolution optical imaging techniques, such asoptical coherence tomography, can image up to approximately onetransport mean free path (about 1 mm) into biological tissue. However,these techniques are insensitive to optical absorption that is relatedto important biochemical information. Other well-known techniques, suchas confocal microscopy and multi-photon microscopy, often involve theintroduction of exogenous dyes, which, with a few notable exceptions,have relatively high toxicity. In addition, acoustic microscopic imagingand spectroscopy systems are sensitive to acoustic impedance variationsonly, which have low contrast for early-stage cancer and provide littlefunctional information about biological tissue except flow. In contrast,photoacoustic wave magnitude is, within certain bounds, linearlyproportional to the optical absorption contrast; thus photoacousticspectral measurement can be performed to gain functional (physiological)information such as the local blood oxygenation level.

BRIEF DESCRIPTION

In one aspect, an imaging method is provided the includes receiving afirst light beam from a light source; splitting the first light beaminto a plurality of second light beams using a beam-divider; focusingthe plurality of second light beams on respective locations in an objectof interest using a focusing device; and receiving the acoustic signalsfrom the object of interest using an ultrasonic transducer array. Theplurality of second light beams may cause the object of interest to emitacoustic signals. The plurality of second light beams and acousticsignals may be coaxially aligned on opposite sides of the object ofinterest in a transmission mode. The method may further includecoaxially merging the plurality of second light beams and the acousticsignals using an optical-acoustic beam combiner. Coaxially merging theplurality of second light beams and the acoustic signals may includepassing the plurality of second light beams through the optical-acousticbeam combiner to the focusing device and reflecting the acoustic signalsentering the optical-acoustic beam combiner toward the ultrasonictransducer array. Coaxially merging the plurality of second light beamsand the acoustic signals may include reflecting the plurality of secondlight beams entering the optical-acoustic beam combiner toward thefocusing device and passing the acoustic signals through theoptical-acoustic beam combiner to the ultrasonic transducer array. Themethod may further include reflecting the first light beam toward thebeam-divider using a movable scanning mirror. The first light beam maybe reflected in a raster scanning pattern. The method may furtherinclude generating an image based on the acoustic signals. The pluralityof second light beams may be formed as a linear array. The acousticsignals may be received by a linear ultrasonic transducer array. Theplurality of second light beams may be formed as a 2D array. Theacoustic signals may be received by a 2D ultrasonic transducer array.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments described herein may be better understood by referringto the following description in conjunction with the accompanyingdrawings.

FIG. 1 is a diagram of a photoacoustic probe of an imaging system inaccordance with one embodiment of the present disclosure, where a 2Dtransmission grating and an optically transparent acoustic reflector areemployed.

FIG. 2 is a block diagram showing the overarching architecture of thepresent disclosure.

FIG. 3 is a diagram of the integrated focusing assembly of an imagingsystem in accordance with an alternative embodiment of the presentdisclosure, where a 2D transmission grating and a custom-builtoptical-acoustic beam combiner are employed.

FIG. 4 is a diagram of the integrated focusing assembly of an imagingsystem in accordance with another alternative embodiment of the presentdisclosure, where a 2D transmission grating and an acousticallytransparent thin-film optical reflector are employed.

FIG. 5 is a diagram of the integrated focusing assembly of an imagingsystem in accordance with another alternative embodiment of the presentdisclosure, where a 2D transmission grating and a transmission-modedesign are employed.

FIG. 6 is a diagram of the photoacoustic probe of an imaging system inaccordance with another alternative embodiment of the presentdisclosure, where a 2D microlens array and an optically transparentacoustic reflector are employed.

FIG. 7 is a diagram of the integrated focusing assembly of an imagingsystem in accordance with yet another embodiment of the presentdisclosure, where a 2D microlens array and a custom-builtoptical-acoustic beam combiner are employed.

FIG. 8 is a diagram of the integrated focusing assembly of an imagingsystem in accordance with yet another embodiment of the presentdisclosure, where a 2D microlens array and an acoustically transparentthin-film optical reflector are employed.

FIG. 9 is a diagram of the integrated focusing assembly of an imagingsystem in accordance with yet another embodiment of the presentdisclosure, where a 2D microlens array and a transmission-mode designare employed.

FIG. 10A shows a photoacoustic image of two crossed 6-micrometerdiameter carbon fibers acquired with the current prototype of thepresent disclosure.

FIG. 10B shows a distribution of the photoacoustic amplitude across thevertical fiber shown in FIG. 10A.

FIGS. 11A and 11B show in vivo photoacoustic images of a mouse earvasculature acquired with the current prototype of the presentdisclosure.

FIG. 12 is a flowchart illustrating an exemplary imaging method.

DETAILED DESCRIPTION

While the making and using of various embodiments of the presentdisclosure are discussed in detail below, it should be appreciated thatthe present disclosure provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the disclosure and do not delimit the scope of thedisclosure.

To facilitate the understanding of this disclosure, a number of termsare defined below. Terms defined herein have meanings as commonlyunderstood by a person of ordinary skill in the areas relevant to thepresent disclosure. Terms such as “a,” “an” and “the” are not intendedto refer to only a singular entity, but include the general class ofwhich a specific example may be used for illustration. The terminologyherein is used to describe specific embodiments of the disclosure, buttheir usage does not delimit the disclosure, except as outlined in theclaims.

To be consistent with the commonly used terminology, whenever possible,the terms used herein will follow the definitions recommended by theOptical Society of America (OCIS codes).

In some embodiments, the term “photoacoustic microscopy” refers to aphotoacoustic imaging technology that detects pressure waves generatedby light absorption in the volume of a material (such as biologicaltissue) and propagated to the surface of the material. In other words,photoacoustic microscopy is a method for obtaining images of the opticalcontrast of a material by detecting acoustic or pressure waves travelingfrom the object. As used herein, the term “photoacoustic microscopy”includes detection of the pressure waves that are still within theobject.

In some embodiments, the terms “reflection mode” and “transmission mode”refer to a laser photoacoustic microscopy system that employs thedetection of acoustic or pressure waves transmitted from the volume oftheir generation to the optically irradiated surface and a surface thatis opposite to, or substantially different from, the irradiated surface,respectively.

In some embodiments, the term “multi-focus 1D array illumination” refersto optical illumination for photoacoustic excitation with aone-dimensional array of focused pulsed laser beams.

In some embodiments, the term “multi-focus matrix illumination” refersto optical illumination for photoacoustic excitation using atwo-dimensional array (matrix) of focused pulsed laser beams.

In some embodiments, the term “linear ultrasonic array” refers to aone-dimensional array of ultrasonic transducers, with which thetwo-dimensional (2D) in-plane spatial distributions and strength ofultrasonic (photoacoustic) sources can be reconstructed based on thetime-resolved signals arriving at the array.

In some embodiments, the term “matrix ultrasonic array” refers to atwo-dimensional array of ultrasonic transducers, with which the 3Dspatial distributions and strength of photoacoustic sources can bereconstructed based on the time-resolved signals arriving at the array.Ultrasonic transducers generally refer to all types of ultrasonic wavedetection devices including devices utilizing optical interferometers tosense ultrasonic waves.

In some embodiments, the term “diffraction limited resolution” refers tothe best possible resolution by focusing light within the limitationsimposed by diffraction.

In some embodiments, the term “photoacoustic emissions” refers to thepressure waves produced by light absorption.

In some embodiments, the term “B-scan image” refers to a cross-sectionaltwo-dimensional image in the plane containing the acoustic axis.

In some embodiments, the term “integrated focusing assembly” refers toan integrated assembly including optical focusing components, anultrasonic array, and the coupling devices between them.

In some embodiments, the term “photoacoustic reconstruction” refers to asignal processing technique used to reconstruct a photoacoustic B-scanimage from received signals.

Embodiments of the present disclosure provide methods, systems, andapparatus for high-speed, optical-resolution photoacoustic imaging usingmulti-focus optical illumination in conjunction with ultrasonic arraydetection. Specifically, embodiments of the present disclosure usemultiple focused pulsed laser beams to produce a rapid local temperaturerise at multiple optical foci from absorption of the pulsed light. Thetemperature rise leads to a transient thermal expansion, resulting inphotoacoustic emissions, which are detected by a high-frequencyultrasonic array to reconstruct an image. The image signal amplitude isrelated to the optical absorption and Grueneisen parameter. With eachlaser pulse, the multi-focus illumination excites photoacoustic wavesfrom multiple sites, which the ultrasonic array detects simultaneously.The use of the ultrasonic array with photoacoustic reconstruction allowsthe separation of signals from the multiple optical illumination sitesas close as the lateral resolution of the ultrasonic array.Fundamentally different from simply combining multiple assemblies of asingle optical focusing element and a single ultrasonic transducer, thisapproach has enabled us to position optical foci much closer to eachother.

Compared with previously existing optical-resolution photoacousticmicroscopy, embodiments of the present disclosure significantly reducethe range or area of scanning for 2D or 3D imaging, respectively, by upto two to three orders, depending on the number of illumination spots.As a result, rapid scanning can be used to produce 2D or 3Dphotoacoustic imaging with optical resolution at high speed—even in realtime. For example, using multi-focus matrix illumination in conjunctionwith matrix ultrasonic array detection, a 3D photoacoustic image can beproduced by rapidly scanning the illumination over a small areacomparable in size with the lateral resolution of the ultrasonic array.In fact, even with a simplified design using 1D array illumination inconjunction with linear ultrasonic array detection, the multi-focusoptical-resolution photoacoustic microscopy device demonstrates asignificant improvement in imaging speed over previously existingoptical-resolution photoacoustic microscopy devices.

At an ultrasonic frequency suitable for ˜1 millimeter (mm) penetrationin soft tissue, the optical diffraction limited spatial resolution canbe two orders of magnitude finer than the acoustically definedcounterpart. In addition, in comparison with a conventional broad lightillumination, the confined illumination significantly reduces thebackground arising from the interference of photoacoustic waves fromvarious targets within the illumination volume. Due to the minimalscanning requirement of the present disclosure, a handheld 3D imagingdevice can also be made, which could be particularly useful for clinicalapplications such as intraoperative surgery. Moreover, the high imagingspeed of the present disclosure is critical for clinical practice inorder to reduce motion artifacts, patient discomfort, cost, and risksassociated with minimally invasive procedures such as endoscopy.

The embodiments described in detail herein employ a tunable dye laserpumped by an Nd:YLF laser as the irradiation source. The laser pulseduration is on the order of several nanoseconds. The pulse repetitionrate, which is controlled by an external triggering signal, can be ashigh as a few kilohertz (kHz), without significant degradation of theoutput energy. In other embodiments, a plurality of sources ofpenetrating radiation, which can be confined to or concentrated in asmall volume within the object, can be used. Such sources include, butare not limited to, pulsed lasers, flash lamps, other pulsedelectromagnetic sources, particle beams, or their intensity-modulatedcontinuous-wave counterparts.

To provide multi-focus optical illumination for photoacousticexcitation, the embodiments described in detail herein use either amicrolens array or a transmission grating in conjunction with anobjective lens; for photoacoustic signal detection, an ultrasonic arrayis used. However, the present disclosure includes any realization oflight focusing using any kind of mirrors, lenses, fibers, and/ordiaphragms that can produce multi-focus illumination confined to thefield of view of a linear or matrix ultrasonic array. In scatteringbiological tissue, the system can image ˜1 mm deep, with axial andlateral resolutions determined by the ultrasonic bandwidth and theoptical focusing, respectively. With an oscillating mirror, the presentdisclosure provides rapid optical scanning for 3D imaging, enablingoptical-resolution photoacoustic microscopy at high speed.

The imaging procedure described herein is one of the possibleembodiments specifically aimed at medical and biological applications.The optical absorption contrast of the present disclosure iscomplementary to other contrasts that can be obtained from purelyoptical or ultrasonic imaging technologies, and can be used fordiagnostic, monitoring, or research purposes. The main applications ofthe technology include, but are not limited to, the imaging of arteries,veins, and pigmented tumors (such as melanomas) in vivo in humans oranimals. The present disclosure can use the spectral properties ofintrinsic optical contrast to monitor blood oxygenation, blood volume(total hemoglobin concentration), and even the metabolic rate of oxygen;it can also use the spectral properties of a variety of dyes or othercontrast agents to obtain additional functional or molecularinformation. In short, the present disclosure is capable of functionaland molecular imaging. In addition, the present disclosure can be usedto monitor possible tissue changes during x-ray radiation therapy,chemotherapy, or other treatment; it can also be used to monitor topicalapplication of cosmetics, skin creams, sun-blocks, or other skintreatment products.

To translate photoacoustic imaging into clinical practice, a highimaging speed is needed to reduce motion artifacts, cost, patientdiscomfort, and most important, the risks associated with minimallyinvasive procedures (e.g., endoscopy). Embodiments described hereinprovide the combined use of multi-focus optical illumination andultrasonic array detection can help photoacoustic imaging meet thechallenges of clinical translation. In addition, embodiments of thepresent disclosure uses tightly focused laser illumination to provideoptical diffraction limited lateral resolution, which is difficult toachieve using ultrasonic approaches. Therefore, embodiments of thepresent disclosure offer a method, apparatus, and system ofphotoacoustic imaging with high imaging speed and spatial resolutionsufficient for many clinical and preclinical applications.

FIG. 1 is a schematic of the photoacoustic probe of an imaging system100 in accordance with one embodiment of the present disclosure, where abeam-divider, such as a 2D transmission grating, and an opticallytransparent acoustic reflector are employed. The light from a wavelengthtunable laser is focused by a condenser lens 101 onto a pinhole 102 forspatial filtering. While a photo-detector 104 is used to monitor thelaser pulse energy through a sampling beam splitter 103, an eyepiece 114is used to optically image the object's surface for alignment. Toprovide multi-focus matrix illumination for photoacoustic excitation,the laser beam from the pinhole is collimated by a collimating lens 105,split by a 2D transmission grating 107, and then tightly focused by afocusing device, such as an objective lens 108, into an imaging object111, after passing through an optically transparent acoustic reflector110. Through an acoustic coupling medium 109 (e.g., ultrasound couplinggel), the photoacoustic signal emitted by the object is reflected by theacoustic reflector and detected by a matrix ultrasonic transducer array112, 113. Inset A of FIG. 1 shows one possible relative positioning forthe focused optical spots and the matrix ultrasonic array elements. Thisdesign is suitable for integration with commercially availableultrasonic transducer arrays (1D or 2D).

FIG. 2 is a block diagram showing the overarching architecture of thepresent disclosure. Components include a high-repetition-rate tunablepulsed laser system, an optical assembly, an ultrasonic array, ahigh-speed multi-channel data acquisition (DAQ) subsystem, a scanningmirror or linear scanner, and a multi-core computer. The opticalassembly receives pulsed laser light and provides multi-focusillumination for photoacoustic excitation. The DAQ system records anddigitizes the received photoacoustic signal. The laser pulse generation,data acquisition, and scanning of the optical illumination aresynchronized using triggering signals from the DAQ card. To optimize thedata acquisition and imaging speed, the number of data acquisitionchannels should match the number of elements of the ultrasonic array.However, when the number of array elements is greater, multiplexers maybe used. An off-the-shelf multi-core computer, together with a parallelcomputing program based on Microsoft Visual Studio or other softwaredevelopment tools, is used to perform photoacoustic reconstruction forhigh-speed imaging and display.

To integrate the optical focusing and the ultrasonic detection for thepresent disclosure, one or more of the following devices or designs canbe used: (1) an optically transparent acoustic reflector; (2) anacoustically transparent optical reflector; and/or (3) directintegration in transmission mode. Examples of the integrated focusingassembly are described with reference to FIGS. 3, 4, 5, 6, 7, 8, and 9,wherein the integrated focusing assembly includes optical focusingcomponents, a matrix ultrasonic array, and an optical-acoustic beamcombiner between them.

FIG. 3 shows the integrated focusing assembly of an imaging system inaccordance with another embodiment of the present disclosure, where a 2Dtransmission grating and a custom-built optical-acoustic beam combinerare employed. In this embodiment, a scanning device, such as a scanningmirror 301, is used for rapid optical scanning; the 2D transmissiongrating 302 is used to split the laser beams; and the optical-acousticbeam combiner 304, 305, 306 is used to merge the optical illuminationand the ultrasonic detection coaxially. The optical-acoustic beamcombiner mainly consists of an isosceles triangular prism 304 and arhomboidal prism 306 (the two prisms are adjoined along the diagonalsurfaces with a gap of approximately 0.1 mm in between). The gap isfilled with an optical refractive-index-matching,low-acoustic-impedance, nonvolatile liquid 305 (e.g., 1000 cSt siliconeoil). The silicone oil and the glass have a good optical refractiveindex match (glass: 1.5; silicone oil: 1.4) but a large acousticimpedance mismatch (glass: 12.1×10⁶ N·s/m³; silicone oil: 0.95×10⁶N·s/m³). As a result, the silicone oil layer is optically transparentbut acoustically reflective. The laser beams coming from the grating aretightly focused by an objective lens 303 into an imaging object 308.Through an acoustic coupling medium 307, the photoacoustic signalemitted from the object is detected by a matrix ultrasonic array 309,310. Note that within the bandwidth of the ultrasonic array, ultrasonicabsorption in the silicone oil is high enough to dampen acousticreverberations in the matching layer and thus minimize interference withthe image.

FIG. 4 shows the integrated focusing assembly of an imaging system inaccordance with yet another embodiment of the present disclosure, wherea 2D transmission grating and an acoustically transparent thin-filmoptical reflector are employed. In this embodiment, a scanning mirror401 is used for rapid optical scanning; the 2D transmission grating 402is used to split the laser beams; and the acoustically transparentoptical reflector 405 (e.g., an aluminized Mylar thin film) is used tomerge the optical illumination and the ultrasonic detection coaxially.The laser beams coming from the grating are tightly focused by anobjective lens 403 into an imaging object 406. Through an acousticcoupling medium 404, the photoacoustic signal emitted from the object isdetected by a matrix ultrasonic array 407, 408.

FIG. 5 shows the integrated focusing assembly of an imaging system inaccordance with yet another embodiment of the present disclosure, wherea 2D transmission grating and a transmission-mode design are employed.In this embodiment, a scanning mirror 501 is used for rapid opticalscanning; the 2D transmission grating 502 is used to split the laserbeams; and the transmission-mode design is used to merge the opticalillumination and the ultrasonic detection coaxially. The laser beamscoming from the grating are tightly focused by an objective lens 503into an imaging object 504. From the other side of the object, through acoupling medium 505, the photoacoustic signal emitted from the object isdetected by a matrix ultrasonic array 506, 507.

FIG. 6 shows the photoacoustic probe of an imaging system in accordancewith yet another embodiment of the present disclosure, where a 2Dmicrolens array and an optically transparent acoustic reflector areemployed. The light from a wavelength tunable laser is focused by acondenser lens 601 onto a pinhole 602 for spatial filtering. Aphoto-detector 604 is used to monitor the laser pulse energy through asampling beam splitter 603, and an eyepiece 612 is used to opticallyimage the object's surface for alignment. To provide multi-focus matrixillumination for photoacoustic excitation, the laser beam from thepinhole is first collimated by a collimating lens 605, and then splitand focused by the 2D microlens array 606 into an imaging object 609,after passing through the optically transparent acoustic reflector 608.Through an acoustic coupling medium 607, the photoacoustic signalemitted from the object is reflected by the acoustic reflector anddetected by a matrix ultrasonic array 610, 611. Inset B of FIG. 6 showsone possible relative positioning for the 2D microlens array and thematrix ultrasonic array. To produce a 3D image, raster scanning of theoptical illumination is required, which, however, can be very fast, dueto the small scanning area.

FIG. 7 shows the integrated focusing assembly of an imaging system inaccordance with yet another embodiment of the present disclosure, wherea 2D microlens array and a custom-built optical-acoustic beam combinerare employed. To provide multi-focus matrix illumination forphotoacoustic excitation, the laser beam from a pinhole 701 is firstcollimated by a collimating lens 702, and then split and focused by the2D microlens array 703 into an imaging object 708, after passing throughthe optical-acoustic beam combiner 704, 705, 706. The optical-acousticbeam combiner mainly consists of an isosceles triangular prism 704 and arhomboidal prism 706 (the two prisms are adjoined along the diagonalsurfaces with a gap of approximately 0.1 mm in between). The gap isfilled with an optical refractive-index-matching,low-acoustic-impedance, nonvolatile liquid 705 (e.g., 1000 cSt siliconeoil). The silicone oil and the glass have a good optical refractiveindex match (glass: 1.5; silicone oil: 1.4) but a large acousticimpedance mismatch (glass: 12.1×10⁶ N·s/m³; silicone oil: 0.95×10⁶N·s/m³). As a result, the silicone oil layer is optically transparentbut acoustically reflective. Through an acoustic coupling medium 707,the photoacoustic signal emitted from the object is detected by a matrixultrasonic array 709, 710. To produce a 3D image, raster scanning of theoptical illumination is required, which, however, can be very fast, dueto the small scanning area.

FIG. 8 shows the integrated focusing assembly of an imaging system inaccordance with yet another embodiment of the present disclosure, wherea 2D microlens array and an acoustically transparent thin-film opticalreflector are employed. To provide multi-focus matrix illumination forphotoacoustic excitation, the laser beam from a pinhole 801 iscollimated by a collimating lens 802, reflected by a dielectric mirror803, and then split and focused by the 2D microlens array 804 into animaging object 807. The acoustically transparent optical reflector 806(e.g., an aluminized Mylar thin film) is used to merge the opticalillumination and the ultrasonic detection coaxially. Through an acousticcoupling medium 805, the photoacoustic signal emitted from the object isdetected by a matrix ultrasonic array 808, 809. To produce a 3D image,raster scanning of the optical illumination is required, which, however,can be very fast, due to the small scanning area.

FIG. 9 shows the integrated focusing assembly of an imaging system inaccordance with yet another embodiment of the present disclosure, wherea 2D microlens array and a transmission-mode design are employed. Thetransmission-mode design is used to merge the optical illumination andthe ultrasonic detection coaxially. To provide multi-focus matrixillumination for photoacoustic excitation, the laser beam from a pinhole901 is first collimated by a collimating lens 902, and then split andfocused by the 2D microlens array 903 into an imaging object 904. Fromthe other side of the object, through a coupling medium 905, thephotoacoustic signal emitted from the object is detected by a matrixultrasonic array 906, 907. To produce a 3D image, raster scanning of theoptical illumination is required, which, however, can be very fast, dueto the small scanning area.

The embodiments described above are based on multi-focus matrixillumination in conjunction with matrix ultrasonic array detection.However, as mentioned above, the design can be simplified to 1D, usingmulti-focus 1D array illumination in conjunction with linear ultrasonicarray detection. With optical scanning, this simplified design canprovide real-time photoacoustic B-scan imaging, sufficient for manybiomedical applications. By adding one additional mechanical scanning,3D images can be produced at a significantly higher speed compared withmechanical scanning single-focus optical-resolution photoacousticmicroscopy. In addition, the implementation of this simplified versionof the present disclosure can be relatively easy and inexpensive.

The above-described embodiments have been successfully demonstrated forbiomedical applications. FIG. 8 illustrates a system prototype based onthe transmission mode design, and using multi-focus 1D arrayillumination in conjunction with linear ultrasonic array detection. Thesystem employs a tunable dye laser pumped by an Nd:YLF laser as theirradiation source. The laser pulse duration is approximately 7nanoseconds (ns), and the pulse repetition rate can be as high as 1.5kHz without significant degradation of the output energy. Forphotoacoustic excitation, a 1D microlens array consisting of 20micro-lenses with a center-to-center spacing of approximately 250micrometers is used, which provides twenty focused illumination spotssimultaneously. For photoacoustic signal detection, a 30-MHz linearultrasonic array consisting of forty-eight elements with a spacing ofapproximately 100 micrometers is used. The system can imageapproximately 1 mm deep in scattering biological tissue, with finer than10-micrometer lateral resolution and an approximately 25-micrometeraxial resolution. Currently, the system acquires a volume image data setof 1000 by 500 by 200 voxels in approximately 4 min, which is about 4times faster than existing mechanical scanning single-focusphotoacoustic microscopy with optical resolution. In principle, using a48-channel DAQ system to eliminate the 6:1 multiplexing, the system canimage about 20 times faster, as determined primarily by the number ofoptical foci. With an increased laser repetition rate and more denselypacked micro-lens array, the imaging speed of the system can be furtherimproved. Of note, the 10-micrometer optically defined lateralresolution is one to two orders of magnitude finer than the acousticallydefined counterpart, enabling the system to image capillary-levelmicrovessels in vivo.

FIG. 10A shows a photoacoustic maximum amplitude projection image of twocrossed 6-micrometer diameter carbon fibers acquired at 570 nm. Maximumamplitude projection refers to projection of the maximum photoacousticamplitudes along a direction—usually the depth or z axis directionunless otherwise mentioned—to its orthogonal plane. FIG. 10B shows thedistribution of the photoacoustic amplitude across the imaged verticalfiber (along the dashed line in FIG. 10A), demonstrating that thelateral resolution of the system is at least as fine as 10 micrometers.

FIGS. 11A and 11B show a photoacoustic maximum amplitude projection anda 3D image of a mouse ear microvasculature, respectively, acquirednoninvasively in vivo. Microvessels, including those in thecapillary-level (less than approximately 10 micrometers), are clearlyimaged. With high imaging speed and optically defined spatialresolution, the preliminary results demonstrate the potential of thepresent disclosure for broad biomedical applications. For example,imaging speed is one critical issue in advancing photoacoustic endoscopyinto clinical practice for early cancer detection or intravascularatherosclerosis imaging. In addition, the high-speed, high-resolutioncapability will open up new possibilities for the study of tumorangiogenesis, diabetes-induced vascular complications, andpharmacokinetics.

FIG. 12 is a flowchart 1200 that illustrates an exemplary imagingmethod. In some embodiments, a scanning device, such as scanning mirror301, a galvanometer, a polygon scanner, an acoustic-optical scanner, andan electro-optical scanner, or another suitable scanning device,receives 1202 a first light beam from a light source, such as a laser orlaser system. A beam-divider, such as transmission grating 107 or 2Dmicrolens array 606, splits 1204 the first light beam into a pluralityof second light beams. A focusing device, such as objective lens 108 orcollimating lens 605, focuses 1206 the second light beams on respectivelocations in object of interest 111. In response to the second lightbeams, the temperature within object 111 at each location rises, whichleads to a transient thermal expansion, resulting in emission ofphotoacoustic signals by object 111. The photoacoustic signals arereceived 1208 by ultrasonic transducer array 112, for example.

In some embodiments, a computer generates an image based on thephotoacoustic signals and displays the image to an operator. Moreover,in some embodiments, the second light beams and the photoacousticsignals are coaxially merged by optical-acoustic beam combiner 304, 305,306 that includes a first prism, such as isosceles triangular prism 304,and a second prism, such as rhomboidal prism 306, separated from firstprism 304 by a gap filled with non-volatile liquid 305.

Embodiments described herein provide methods, systems, and apparatus forhigh-speed, optical-resolution photoacoustic imaging using multi-focusoptical illumination in conjunction with ultrasonic array detection. Forexample, embodiments of the present disclosure use multiple focusedpulsed laser beams to produce a rapid local temperature rise at multipleoptical foci from absorption of the pulsed light. The temperature riseleads to a transient thermal expansion, resulting in photoacousticemissions, which are detected by a high-frequency ultrasonic array toreconstruct an image. The image signal amplitude is related to theoptical absorption and the Grueneisen parameter. With each laser pulse,the multi-focus illumination excites photoacoustic waves from multiplesites, which the ultrasonic array detects simultaneously. With anappropriate reconstruction algorithm, the signals from the multiplesites are separated. Using multi-focus matrix illumination and matrixultrasonic array detection, a three-dimensional (3D) photoacoustic imagecan be produced by rapidly scanning the illumination over a small areacomparable in size with the lateral resolution of the ultrasonic array.Consequently, a relatively large 3D volume (e.g., 10 mm×10 mm×1 mm) canbe imaged at high speed—even in real time—with optical diffractionlimited lateral resolution (e.g., 5 micrometers). In one embodiment ofthe present disclosure, multi-focus one-dimensional (1D) arrayillumination and linear ultrasonic array detection can be used. Evenwith this simplified design, the multi-focus optical-resolutionphotoacoustic microscopy device demonstrates a significant improvementin imaging speed over previous optical-resolution photoacousticmicroscopy devices. Overall, embodiments of the present disclosureprovide 3D photoacoustic microscopy of optical absorption contrast withoptical diffraction limited lateral resolution at high speed.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the disclosure.The principal features of this disclosure can be employed in variousembodiments without departing from the scope of the disclosure. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this disclosure and are covered by the claims.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of thisdisclosure have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations can beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the method described herein without departing fromthe concept, spirit and scope of the disclosure. All such similarsubstitutes and modifications apparent to those skilled in the art aredeemed to be within the spirit, scope and concept of the disclosure asdefined by the appended claims

It will be understood by those of skill in the art that information andsignals may be represented using any of a variety of differenttechnologies and techniques (e.g., data, instructions, commands,information, signals, bits, symbols, and chips may be represented byvoltages, currents, electromagnetic waves, magnetic fields or particles,optical fields or particles, or any combination thereof). Likewise, thevarious illustrative logical blocks, modules, circuits, and algorithmsteps described herein may be implemented as electronic hardware,computer software, or combinations of both, depending on the applicationand functionality. Moreover, the various logical blocks, modules, andcircuits described herein may be implemented or performed with a generalpurpose processor (e.g., microprocessor, conventional processor,controller, microcontroller, state machine or combination of computingdevices), a digital signal processor (“DSP”), an application specificintegrated circuit (“ASIC”), a field programmable gate array (“FPGA”) orother programmable logic device, discrete gate or transistor logic,discrete hardware components, or any combination thereof designed toperform the functions described herein. Similarly, steps of a method orprocess described herein may be embodied directly in hardware, in asoftware module executed by a processor, or in a combination of the two.A software module may reside in RAM memory, flash memory, ROM memory,EPROM memory, EEPROM memory, registers, hard disk, a removable disk, aCD-ROM, or any other form of storage medium known in the art. Althoughpreferred embodiments of the present disclosure have been described indetail, it will be understood by those skilled in the art that variousmodifications can be made therein without departing from the spirit andscope of the disclosure as set forth in the appended claims.

A controller, computer, or computing device, such as those describedherein, includes at least one processor or processing unit and a systemmemory. The controller typically has at least some form of computerreadable media. By way of example and not limitation, computer readablemedia include computer storage media and communication media. Computerstorage media include volatile and nonvolatile, removable andnon-removable media implemented in any method or technology for storageof information such as computer readable instructions, data structures,program modules, or other data. Communication media typically embodycomputer readable instructions, data structures, program modules, orother data in a modulated data signal such as a carrier wave or othertransport mechanism and include any information delivery media. Thoseskilled in the art are familiar with the modulated data signal, whichhas one or more of its characteristics set or changed in such a manneras to encode information in the signal. Combinations of any of the aboveare also included within the scope of computer readable media.

Although the present disclosure is described in connection with anexemplary imaging system environment, embodiments of the disclosure areoperational with numerous other general purpose or special purposeimaging system environments or configurations. The imaging systemenvironment is not intended to suggest any limitation as to the scope ofuse or functionality of any aspect of the disclosure. Moreover, theimaging system environment should not be interpreted as having anydependency or requirement relating to any one or combination ofcomponents illustrated in the exemplary operating environment.

Embodiments of the disclosure may be described in the general context ofcomputer-executable instructions, such as program components or modules,executed by one or more computers or other devices. Aspects of thedisclosure may be implemented with any number and organization ofcomponents or modules. For example, aspects of the disclosure are notlimited to the specific computer-executable instructions or the specificcomponents or modules illustrated in the figures and described herein.Alternative embodiments of the disclosure may include differentcomputer-executable instructions or components having more or lessfunctionality than illustrated and described herein.

When introducing elements of aspects of the disclosure or embodimentsthereof, the articles “a,” “an,” “the,” and “said” are intended to meanthat there are one or more of the elements. The terms “comprising,”including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements.

This written description uses examples to disclose the disclosure,including the best mode, and also to enable any person skilled in theart to practice the disclosure, including making and using any devicesor systems and performing any incorporated methods. The patentable scopeof the disclosure is defined by the claims, and may include otherexamples that occur to those skilled in the art. Such other examples areintended to be within the scope of the claims if they have structuralelements that do not differ from the literal language of the claims, orif they include equivalent structural elements with insubstantialdifferences from the literal language of the claims.

What is claimed is:
 1. An imaging method comprising: receiving a firstlight beam from a light source; splitting the first light beam into aplurality of second light beams using a beam-divider; focusing theplurality of second light beams on a respective plurality of locationsin an object of interest using a focusing device, the plurality ofsecond light beams causing the object of interest to emit time-resolvedacoustic signals from the respective plurality of locations; andreceiving the time-resolved acoustic signals from the respectiveplurality of locations in the object of interest using an ultrasonictransducer array, the respective plurality of locations positionedwithin a field of view of the ultrasonic transducer array; wherein theimaging method is an optical-resolution photoacoustic imaging methodwith a lateral resolution of 10 μm or less.
 2. An imaging method inaccordance with claim 1, wherein the plurality of second light beams andthe time-resolved acoustic signals are coaxially aligned on oppositesides of the object of interest in a transmission mode.
 3. An imagingmethod in accordance with claim 1, further comprising coaxially mergingthe plurality of second light beams and the time-resolved acousticsignals using an optical-acoustic beam combiner.
 4. An imaging method inaccordance with claim 3, wherein coaxially merging the plurality ofsecond light beams and the time-resolved acoustic signals comprisespassing the plurality of second light beams through the optical-acousticbeam combiner to the focusing device and reflecting the time-resolvedacoustic signals entering the optical-acoustic beam combiner toward theultrasonic transducer array.
 5. An imaging method in accordance withclaim 3, wherein coaxially merging the plurality of second light beamsand the time-resolved acoustic signals comprises reflecting theplurality of second light beams entering the optical-acoustic beamcombiner toward the focusing device and passing the time-resolvedacoustic signals through the optical-acoustic beam combiner to theultrasonic transducer array.
 6. An imaging method in accordance withclaim 1, further comprising reflecting the first light beam toward thebeam-divider using a movable scanning mirror.
 7. An imaging method inaccordance with claim 6, wherein the first light beam is reflected in araster scanning pattern.
 8. An imaging method in accordance with claim1, further comprising generating an image based on the time-resolvedacoustic signals.
 9. An imaging method in accordance with claim 1,wherein the plurality of second light beams are formed as a lineararray.
 10. An imaging method in accordance with claim 9, wherein thetime-resolved acoustic signals are received by a linear ultrasonictransducer array.
 11. An imaging method in accordance with claim 1,wherein the plurality of second light beams are formed as a 2D array.12. An imaging method in accordance with claim 11, wherein thetime-resolved acoustic signals are received by a 2D ultrasonictransducer array.